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Appl. Sci. 2020, 10, 4290 4 of 9 2.5. Cytotoxicity Assay Primary human gingival fibroblast (HuGu) cell obtained from the Iranian Biologic Resource Center (Tehran, Iran) was cultured in Dulbecco’s modified Eagle’s medium (DMEM, HiMedia Labs, India) supplemented with 10% fetal bovine serum (FBS; Sigma, USA), L-glutamine (2 mM), 1% penicillin/streptomycin antibiotic solution (10,000 Unit/mL penicillin and 10-mg/mL streptomycin) and 100 g/mL of amphotericin B. After incubation at 37 ◦C in a humidified atmosphere of 95% air and 5% CO2, 5 × 103 cells per well were placed in flat-bottom 96-well cell culture microplate and incubated for 24 h at 37 ◦C in a humidified CO2 incubator. Subsequently, 100 μL of Photoactive+ at final concentration of 0.01 g/mL were added to wells and the microplate was incubated at 37 ◦C for 24 h in a humidified atmosphere of 95% air and 5% CO2. For measuring cell viability, the MTT (3-(4,5-dimethyl-thiazoyl)-2,5-diphenyl-SH-tetrazolium bromide) assay was performed according to published protocol [18] and the absorbance values of each well were read at 570 nm through enzyme-linked immuno-assay (ELISA) plate reader. An inverted microscope (Olympus, India) at magnification of 40× was used to assess the cell morphology before and after treatment. 2.6. Statistical Analysis All experiments were performed in triplicate. Data from the experimental groups were analyzed by using two-way analysis of variance (ANOVA) followed by Tukey’s test. The results were presented as mean ± standard deviation. Significance was defined as p-values < 0.05. 3. Results The E. faecalis count with aPDT treatments using Photoactive+ activated by red wavelengths (635 nm and 660 nm) and combination of LED (420–480 nm) and red wavelengths (635 nm and 660 nm) were significantly reduced compared to the control group in both irradiation time (p < 0.05) (Figure 1). aPDT with LED (420–480 nm) decreased the bacterial count, but with no significant difference from the control group in both energy density of 26.4 and 76 J/cm2 (p = 0.75, p = 0.5). The Photoactive+ without activation reduced the count of bacteria with no significant difference from control group. In addition, all light sources including 635 nm, 660 nm and LED (420–480 nm) showed no significant difference in bacteria reduction compared to control group. The greatest reduction in bacterial count was seen with 635-nm red laser activation for energy density of 76 J/cm2 (83%) and lowest with the Photoactive+ without activation (0.8%). Conversely, considering the energy density, the higher energy density of 76 J/cm2 showed a higher reduction of the bacterial count compared to lower energy density of 26.4 J/cm2 in all groups (Figure 1). The mean and standard deviation of experimental groups were shown in Table 2. Table 2. Mean ± standard deviation of experimental groups. Groups Control Photoactive+ R1 R2 Blue R1 + Photoactive+ R2 + Photoactive+ Blue + Photoactive+ Blue–R1 + Photoactive+ Blue-R2 + Photoactive+ R1–Blue + Photoactive+ R2-Blue + Photoactive+ CFU/mL (Mean ± Standard Deviation) × 105 24.6 J/cm2 13 ± 1.9 12.9 ± 0.49 12.35 ± 0.45 12.2 ± 0.35 12.46 ± 0.59 4.3 ± 0.96 5.9 ± 0.94 12.5 ± 0.8 6 ± 0.58 7.5 ± 1.49 6.3 ± 0.85 7.3 ± 1.49 76 J/cm2 13 ± 1.9 12.9 ± 0.49 11.34 ± 0.3 11.1 ± 0.15 12.1 ± 0.45 2.2 ± 0.48 3.1 ± 0.8 11.6 ± 0.65 3.6 ± 0.77 4.7 ± 1.17 3.3 ± 0.66 4.2 ± 0.66PDF Image | Antimicrobial Photodynamic Therapy Chlorophyllin–Phycocyanin
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